Microfluidic chip-based, universal coagulation assay

ABSTRACT

A microfluidic, chip-based assay device has been developed for measuring physical properties of an analyte (particularly, whole blood or whole blood derivatives). The technologies can be applied to measure clotting times of whole blood or blood derivatives, determine the effects of anticoagulant drugs on the kinetics of clotting/coagulation, as well as evaluate the effect of anticoagulant reversal agents. These technologies can additionally be used to optimize the dosage of anticoagulation drugs and/or their reversal agents. The assay is independent of the presence of anticoagulant; clotting is activated by exposure of the blood sample in the device to a glass (or other negatively charged material such as oxidized silicon) surface, which activates the intrinsic pathway and can be further hastened by the application of shear flow across the activating materials surface. The absence of chemical activating agents and highly controlled and reproducible micro-environment yields a point of care universal clotting assay.

CROSS REFERENCE TO RELATED APPLICATION

This application claims benefit of and priority to U.S. ProvisionalApplication No. 62/048,183, filed Sep. 9, 2014, all of which isincorporated by reference in its entirety.

FIELD OF THE INVENTION

The present invention relates to a point of care microfluidic chip-baseduniversal coagulation assay device and reader that can be used tomeasure the global coagulation status of normal healthy, coagulationimpaired, anticoagulated, and anticoagulant reversed patients.

BACKGROUND OF THE INVENTION

Coagulation (clotting) is the process by which blood changes from aliquid to a gel. It potentially results in hemostasis, the cessation ofblood loss from a damaged vessel, followed by repair. The mechanism ofcoagulation involves activation, adhesion, and aggregation of plateletsalong with conversion of fibrinogen to fibrin, which deposits andmatures into a robust network. Disorders of coagulation are diseasestates which can result in bleeding or obstructive clotting(thrombosis).

Coagulation begins very quickly after an injury to the blood vessel hasdamaged the endothelium lining the vessel. Exposure of blood to thespace under the endothelium initiates two categories of processes:changes in platelets, and the exposure of subendothilial tissue factorto plasma Factor VII, which ultimately leads to fibrin formation.Platelets immediately form a plug at the site of injury; this is calledprimary hemostasis. Secondary hemostasis occurs simultaneously:additional coagulation factors or clotting factors beyond Factor VII,respond in a complex cascade to form fibrin strands, which strengthenthe platelet plug.

The coagulation cascade of secondary hemostasis has two pathways whichlead to fibrin formation. These are the contact activation pathway (alsoknown as the intrinsic pathway), and the tissue factor pathway (alsoknown as the extrinsic pathway). It was previously thought that thecoagulation cascade consisted of two pathways of equal importance joinedto a common pathway. It is now known that the primary pathway for theinitiation of blood coagulation is the tissue factor pathway. Thepathways are a series of reactions, in which a zymogen (inactive enzymeprecursor) of a serine protease and its glycoprotein co-factor areactivated to become active components that then catalyze the nextreaction in the cascade, ultimately resulting in cross-linked fibrin.Coagulation factors are generally indicated by Roman numerals, with alowercase “a” appended to indicate an active form.

The coagulation factors are generally serine proteases which act bycleaving downstream proteins. There are some exceptions. For example,FVIII and FV are glycoproteins, and Factor XIII is a transglutaminase.The coagulation factors circulate as inactive zymogens. The coagulationcascade is classically divided into three pathways. The tissue factorand contact activation pathways both activate the “final common pathway”of factor X, thrombin and fibrin.

Tissue Factor Pathway (Extrinsic)

The main role of the tissue factor pathway is to generate a “thrombinburst”, a process by which thrombin, the most important constituent ofthe coagulation cascade in terms of its feedback activation roles, isreleased very rapidly. FVIIa circulates in a higher amount than anyother activated coagulation factor. The process includes the followingsteps:

Following damage to the blood vessel, FVII leaves the circulation andcomes into contact with tissue factor (TF) expressed ontissue-factor-bearing cells (stromal fibroblasts and leukocytes),forming an activated complex (TF-FVIIa).

TF-FVIIa activates FIX and FX.

FVII is itself activated by thrombin, FXIa, FXII and FXa.

The activation of FX (to form FXa) by TF-FVIIa is almost immediatelyinhibited by tissue factor pathway inhibitor (TFPI).

FXa and its co-factor FVa form the prothrombinase complex, whichactivates prothrombin to thrombin.

Thrombin then activates other components of the coagulation cascade,including FV and FVIII (which activates FXI, which, in turn, activatesFIX), and activates and releases FVIII from being bound to vWF.

FVIIIa is the co-factor of FIXa, and together they form the “tenase”complex, which activates FX; and so the cycle continues. (“Tenase” is acontraction of “ten” and the suffix “-ase” used for enzymes.)

Contact Activation Pathway (Intrinsic)

The contact activation pathway begins with formation of the primarycomplex on collagen by high-molecular-weight kininogen (HMWK),prekallikrein, and FXII (Hageman factor). Prekallikrein is converted tokallikrein and FXII becomes FXIIa. FXIIa converts FXI into FXIa. FactorXIa activates FIX, which with its co-factor FVIIIa form the tenasecomplex, which activates FX to FXa. The minor role that the contactactivation pathway has in initiating clot formation can be illustratedby the fact that patients with severe deficiencies of FXII, HMWK, andprekallikrein do not have a bleeding disorder. Instead, contactactivation system seems to be more involved in inflammation.

Coagulation Assays

Several techniques, including clot-based tests, chromogenic or colorassays, direct chemical measurements, and ELISAs, are used forcoagulation testing. Of these techniques, clot-based and chromogenicassays are used most often. Whereas clotting assays provide a globalassessment of coagulation function, chromogenic tests are designed tomeasure the level or function of specific factors.

Clot-based assays are often used for evaluation of patients withsuspected bleeding abnormalities and to monitor anticoagulant therapy.Most of these tests use citrated plasma, which requires tens of minutesfor preparation and typically requires hours to days to receive resultsin a hospital setting. The end point for most clotting assays is fibrinclot formation.

Prothrombin Time (PT) is performed by adding a thromboplastin reagentthat contains tissue factor (which can be recombinant in origin orderived from an extract of brain, lung, or placenta) and calcium toplasma and measuring the clotting time. The PT varies with reagent andcoagulometer but typically ranges between 10 and 14 seconds. The PT isprolonged with deficiencies of factors VII, X, and V, prothrombin, orfibrinogen and by antibodies directed against these factors. This testalso is abnormal in patients with inhibitors of the fibrinogen-to-fibrinreaction, including high doses of heparin and the presence of fibrindegradation products. Typically, PT reagents contain excess phospholipidso that nonspecific inhibitors (i.e., lupus anticoagulants), which reactwith anionic phospholipids, do not prolong the clotting time. The PT ismost frequently used to monitor warfarin therapy. PT measurements arenot comparable between devices or centers and most warfarin clinicsdevelop their own normal patient range, which is non-transferrable andhighly specific to the exact reagents present in the specific assayused.

The activated Partial Thromboplastin Time (aPTT) assay is performed byfirst adding a surface activator (e.g., kaolin, celite, ellagic acid, orsilica) and diluted phospholipid (e.g., cephalin) to citrated plasma. Atthe point of care, aPTT can also be measured in whole blood typicallyusing similar chemical activating agents. The phospholipid in this assayis called partial thromboplastin because tissue factor is absent. Afterincubation to allow optimal activation of contact factors (factor XII,factor XI, prekallikrein, and high-molecular-weight kininogen), calciumis then added, and the clotting time is measured. aPTT measurements arenot comparable between devices or hospitals and most clinicallaboratories develop their own normal patient range, which isnon-transferrable and highly specific to the exact reagents present inthe specific assay used.

Although the clotting time varies according to the reagent andcoagulometer used, the aPTT typically ranges between 22 and 40 seconds.The aPTT may be prolonged with deficiencies of contact factors; factorsIX, VIII, X, or V; prothrombin; or fibrinogen. Specific factorinhibitors, as well as nonspecific inhibitors, may also prolong theaPTT. Fibrin degradation products and anticoagulants (e.g., heparin,direct thrombin inhibitors, or warfarin) also prolong the aPTT, althoughthe aPTT is less sensitive to warfarin than is the PT.

The thrombin clotting time (TCT) is performed by adding excess thrombinto plasma. The TCT is prolonged in patients with low fibrinogen levelsor dysfibrinogenemia and in those with elevated fibrin degradationproduct levels. These abnormalities are commonly seen with disseminatedintravascular coagulation. The TCT is also prolonged by heparin anddirect thrombin inhibitors.

The activated clotting time (ACT) is a point-of-care whole-bloodclotting test used to monitor high-dose heparin therapy or treatmentwith bivalirudin. The dose of heparin or bivalirudin required in thesesettings is beyond the range that can be measured with the aPTT.Typically, whole blood is collected into a tube or cartridge containinga coagulation activator (e.g., celite, kaolin, or glass particles) and amagnetic stir bar, and the time taken for the blood to clot is thenmeasured. The reference value for the ACT ranges between 70 and 180seconds. The desirable range for anticoagulation depends on theindication and the test method used. The ACT does not correlate wellwith other coagulation tests.

For the ecarin clotting time (ECT), venom from the Echis carinatus snakeis used to convert prothrombin to meizothrombin, a prothrombinintermediate that is sensitive to inhibition by direct thrombininhibitors. The ECT cannot be used to detect states of disturbedcoagulation and is useful only for therapeutic drug monitoring. Thisassay is insensitive to heparin because steric hindrance prevents theheparin-antithrombin complex from inhibiting meizothrombin. Becauseecarin also activates the noncarboxylated prothrombin found in plasma ofwarfarin-treated patients, levels of direct thrombin inhibitors can beassayed even with concomitant warfarin treatment. Although the ECT hasbeen used in preclinical research, the test has yet to be standardizedand is not widely available.

Anti-factor Xa assays are used to measure levels of heparin andlow-molecular-weight heparin (LMWH). These are chromogenic assays thatuse a factor Xa substrate onto which a chromophore has been linked.Factor Xa cleaves the chromogenic substrate, releasing a coloredcompound that can be detected with a spectrophotometer and is directlyproportional to the amount of factor Xa present. When a known amount offactor Xa is added to plasma containing heparin (or LMWH), the heparinenhances factor Xa inhibition by antithrombin rendering less factor Xaavailable to cleave the substrate. By correlating this result with astandard curve produced with known amounts of heparin, we can calculatethe heparin concentration in the plasma. The use of anti-Xa assaysrequires the knowledge of which anticoagulant the patient is taking inorder to use the appropriate calibrator and cannot be used to monitoranti-IIa anticoagulant therapies.

Anticoagulant drugs in clinical use include warfarin, heparins(unfractionated heparin and LMWH), and direct thrombin inhibitors(bivalirudin, hirudin, and argatroban).

Warfarin is effective for primary and secondary prevention of venousthromboembolism; for prevention of cardioembolic events in patients withatrial fibrillation or prosthetic heart valves; for prevention ofstroke, recurrent infarction, or cardiovascular death in patients withacute myocardial infarction; and for the primary prevention of acutemyocardial infarction in high-risk men. Because of the variability inthe anticoagulant response to warfarin, which reflects geneticvariations in metabolism and environmental factors such as medications,diet, and concomitant illness, regular coagulation monitoring and dosageadjustment are required to maintain the International Normalized Ratio(INR) within the therapeutic range. Heparins are indirect anticoagulantsthat activate antithrombin and promote its capacity to inactivatethrombin and factor Xa. To catalyze thrombin inhibition, heparin bindsboth to antithrombin via a high-affinity pentasaccharide sequence and tothrombin. In contrast, to promote factor Xa inhibition, heparin needsonly to bind to antithrombin via its pentasaccharide sequence. Heparinmolecules containing <18 saccharide units are too short to bind to boththrombin and antithrombin and therefore cannot catalyze thrombininhibition. However, these shorter heparin fragments can catalyze factorXa inhibition, provided that they contain the pentasaccharide sequence.The anticoagulant response to heparin is unpredictable because ofvariable nonspecific binding to endothelial cells, monocytes, and plasmaproteins. Because of this variable anticoagulant response, coagulationmonitoring is routinely performed when heparin is given in greater thanprophylactic doses. The aPTT is the test most often used to monitorheparin. Unfortunately, aPTT reagents vary in their responsiveness toheparin, and the aPTT therapeutic range differs, depending on thesensitivity of the reagent and the coagulometer used for the test. TheaPTT has proved more difficult to standardize than the PT, and thecommonly quoted therapeutic range of 1.5 to 2.5 times the control valueoften leads to systematic administration of subtherapeutic heparindoses. The evidence supporting the concept of an aPTT therapeutic rangethat predicts efficacy and safety (with respect to bleeding) is somewhattenuous. Approximately 25% of patients require doses of heparin of >35000 U/d to obtain a therapeutic aPTT and are called heparin resistant.Most of these patients have therapeutic heparin levels when measuredwith the anti-Xa assay, and the discrepancy between the 2 tests is theresult of high concentrations of procoagulants such as fibrinogen andfactor VIII, which shorten the aPTT. Although the aPTT response islinear with heparin levels within the therapeutic range, the aPTTbecomes immeasurable with higher heparin doses. Thus, a less sensitivetest of global anticoagulation such as the ACT is used to monitor thelevel of anticoagulation in patients undergoing percutaneous coronaryinterventions or aortocoronary bypass surgery.

LMWH is derived from unfractionated heparin by chemical or enzymaticdepolymerization. LMWH has gradually replaced heparin for mostindications. LMWH is typically administered in fixed doses when givenfor prophylactic purposes or in weight-adjusted doses when given fortreatment. Pitfalls in the monitoring of LMWH by anti-factor Xa levelsinclude poor comparability between commercially available anti-Xachromogenic assays, differences in ratios of anti-Xa to anti-IIa amongthe various LMWH preparations, and the importance of timing of bloodsampling in relation to dosing. Although the aPTT may be prolonged withhigh doses of LMWH, this assay is not used for monitoring. No clinicallyavailable point of care assay to date is available for the monitoring ofthe millions of patients administered LMWH.

Direct thrombin inhibitors bind directly to thrombin and block theinteraction of thrombin with its substrates. Three parenteral directthrombin inhibitors have been licensed for limited indications in NorthAmerica. Hirudin and argatroban are approved for treatment of patientswith heparin-induced thrombocytopenia, whereas bivalirudin is licensedas an alternative to heparin in patients undergoing percutaneouscoronary intervention (PCI). Hirudin and argatroban require routinemonitoring. The TCT is too sensitive to small amounts of hirudin andargatroban to be used for this purpose. Although the ACT has been usedto monitor the higher doses of direct thrombin inhibitors required ininterventional settings, it does not provide an optimal linear responseat high concentrations. The aPTT is recommended for therapeuticmonitoring; however, each direct thrombin inhibitor has its own doseresponse, and the sensitivity of the test to drug levels varies betweenaPTT reagents. When hirudin therapy is monitored with the aPTT, the doseis adjusted to maintain an aPTT that is 1.5 to 2.5 times the control,whereas for argatroban, the target aPTT is 1.5 to 3 times control (butnot to exceed 100 seconds). The aPTT appears less useful in patientsrequiring higher doses of direct thrombin inhibitor in cardiopulmonarybypass procedures because this test becomes less responsive atincreasing drug concentrations. The ECT appears to be useful for bothlow and high concentrations of direct thrombin inhibitors and is lessaffected by interfering substances than the aPTT. However, as statedabove, it is not routinely available. The responsiveness of the INR todifferent drug concentrations differs with assay reagent and with thetype of direct thrombin inhibitor. This feature complicates thetransitioning of patients with heparin-induced thrombocytopenia fromargatroban to vitamin K antagonists.

As is clear from the foregoing, clotting, and inhibition of clotting, isa complex process. The type of anticoagulant can give misleading anddangerous results if determined using the wrong clotting assay. Thiscreates a potentially disastrous scenario when an anticoagulated patientarrives at an emergency room without information as to medicines he ison, as well as the condition being treated. Sometimes it is impossibleto wait for further diagnostics to determine the anticoagulant ordisorder causing prolonged bleeding. The need for a rapid, accurate, anduniversal test for clotting, especially a point of care (“POC”) test, iswell known; options, however, are extremely limited.

It is therefore an object of the present invention to provide a rapid,accurate and universal test for clotting.

It is a further object of the present invention to provide a point ofcare test for clotting.

It is a still further object of the present invention to provide a testthat is accurate, reproducible, easy to operate, and requires a verysmall amount of sample.

SUMMARY OF THE INVENTION

A microfluidic, chip-based assay device has been developed for measuringphysical properties of an analyte (in particular, whole blood or wholeblood derivatives). The technologies can in particular be applied tomeasure clotting times of whole blood or blood derivatives, to determinethe effects of anticoagulant drugs on the kinetics ofclotting/coagulation, as well as to evaluate the effect of anticoagulantreversal agents. These technologies can additionally be used to optimizethe dosage of anticoagulation drugs and/or their reversal agents. Theassay is independent of the presence of anticoagulant since clotting isactivated by exposure of the blood sample in the device to a glass (orother negatively charged material such as oxidized silicon) surface,which activates the intrinsic pathway and can be further hastened by theapplication of shear flow across the activating materials surface. Theabsence of chemical activating agents and highly controlled andreproducible micro-environment yields a point of care universal clottingassay.

The sample is handled in a microfluidic system. The volume of sampleintroduced into the testing chamber is in the nano-, micro-, ormilliliter range (most preferably 1-10 microliters). The sample isintroduced into the collection well directly from a blood sample or theindividual (such as from a finger stick). In one embodiment, the sample,such as blood or plasma, is collected and transferred to the heatedmicrodevice immediately after collection by syringe into a no-additivered-topped tube or capillary tube. Clotting in the samples, preferablyin duplicate, is initiated by exposure of the blood or plasma to theglass surface within the device and the sample exposed to means foranalysis of clotting. The blood sample is then drawn from the collectionwell into the testing chamber either passively by capillary action orwith a pump, which induces sheer and exposes the blood sample to theactivating materials surface, while delivering a geometricallycontrolled amount of the blood sample into the testing chamber. Themicrofluidic system along with integrated electrodes, heater structuresor other parts of sensors or actuators is designed to be disposable. Themicrofluidic system is inserted into an analytical re-usable housing(referred to herein as a “reader”) that is part of an analysisinstrument, which connects the microfluidic system and provides fluidic,electrical, optical and thermal interfaces for measuring clotting andtransmitting the time and characteristics of the measurement to anexternal reader, monitor, or recorder.

Clotting is assessed by a change in viscosity, optical transmission,electrical impedance, and/or pressure. In the preferred embodiment,clotting is detected through measurement of blood impedance throughintegrated electrodes and/or through measurement of optical transmissionusing infrared (IR) LEDs and photodiodes, respectively. An integratedthermal resistive heater/cooler structure such as a solid state heatpump or Peltier cooler keeps the blood sample at a defined temperature,most preferably approximately 37° C. (body temperature), and ensuresrepeatability and comparability of measurements. As fibrinogen convertsto fibrin, the IR absorbance increases until it peaks in a measurablefashion. The determination of whole blood clotting time is made on orabout the peak of IR absorbance. After completion of measurements, themicrofluidic chip containing the blood sample is removed from the readerand discarded.

The development of clotting can be monitored by measuring electricalimpedance. The development of fibrin increases electrical impedanceuntil it peaks in a measurable fashion. On or about the peak ofelectrical impedance, the determination of whole blood clotting time ismade. Whole blood clotting time can be measured by IR absorption andelectrical impedance measured alone or simultaneously as a basis ofcomparison. The electrical impedance and IR absorption curves areessentially coincident, and provide confirmation via independentmeasurement modes.

The microfluidic system is fabricated through application ofmicrotechnologies and processing and bonding of wafers made of silicon,glass or other suitable materials. The microfluidic system can also befabricated through alternative means, for example, through applicationof soft lithography technologies or through generation of reader parts(made, for example, of plastic or a different suitable, substantiallyIR-opaque material) that may be used alone or in combination withmicropatterned chips to form a microfluidic system. The microfluidicsystem typically consists of an inlet, an outlet and one or morechambers that are connected through channels, which range in length fromtens of microns to millimeters, with heights and depths in the tens ofmicrons to hundreds of microns range.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a schematic of the coagulation cascade.

FIG. 2A is a cross-sectional view of one embodiment of the microfluidicdevice in which blood is pipetted into one chamber, and the chip is theninserted into packaging.

FIG. 2B is a cross-sectional view of an embodiment of the microfluidicdevice where blood can be introduced through chamber side; “sipper”sticks out of reader, similar to blood glucose measurement.

FIG. 3A is a view of the open version of the two sample chamber devices.

FIG. 3B is a view of the closed of the two sample chamber devices.

FIGS. 4A-4H are views of the chambers showing the chambers, connectingchannels, electrical contact pads, and thermistors.

FIG. 5 is a perspective view from the top of the bottom of the closedversion reader.

FIG. 6 is a cross-sectional view of the side of the bottom of the closedversion reader.

FIG. 7 is a perspective cross-sectional view of the top of the closedversion reader showing the sample assaying device in place.

FIG. 8 is a perspective cross-sectional view from the top of the top ofthe closed version reader.

FIG. 9A is a perspective cross-sectional view from the top of the bottompart of the open version reader.

FIG. 9B is a perspective cross-sectional view from the side of thebottom part of the open version reader.

FIG. 10A is a perspective cross-sectional view from the side and bottomof the top of the open version reader.

FIG. 10B is a perspective cross-sectional view from the side and top ofthe top of the open version reader.

FIG. 11 is a schematic of the system, showing a box containing thesingle use disposable assay chambers, the reader, and the connections toa computer processor and monitor.

FIGS. 12A-12F are graphs of the impedance (12A, 12C, and 12E) andinfrared transmission (12B, 12D, 12F) over time in minutes for controlno anticoagulant, 300 ng edoxaban anticoagulant/ml blood, and 300 ngedoxaban and ciraparantag (PER977; an anticoagulant reversal agent)/mlof blood, respectively.

DETAILED DESCRIPTION OF THE INVENTION I. Definitions

Microfluidics

Microfluidics is a highly interdisciplinary field, drawing fromengineering, physics, chemistry, biochemistry, micro-/nanotechnology,and biotechnology. Small volumes of liquid, ranging from femto- tomilliliters are typically handled in a microfluidic system. The methodsfor fabrication of a microfluidic system typically allow for integrationof sensors and/or actuators, so that liquids can be effectivelytransported, manipulated and analyzed inside the microfluidic system.Interfaces between the microfluidic system and its environment enableimplementation of external mechanisms for transport, manipulation andanalysis.

Micro-/Nanotechnologies

Micro-/Nanotechnologies are typically used to fabricate microsystems,including microfluidic systems. Micro-/nanotechnologies typically enablegeneration of structures with dimensions in the micro—or nanometerrange. Such technologies can be based on silicon wafer processingtechnologies, originally developed for fabrication of integratedelectronic circuits.

Capillary Action

A convenient way of loading a liquid sample into a microfluidic systemis by capillary action. A sample collection port is wetted with thesample and the sample is effectively drawn into the narrow, hydrophilicchannels and chambers of the microfluidic system by capillary forces.

Anticoagulant

An anticoagulant is a substance that interferes with the ability ofblood to clot. Administered as a therapeutic drug, an anticoagulant can,for example, help reduce or prevent the occurrence of potentiallyhealth- and/or life-threatening emboli or thrombi.

Anticoagulant Reversal Agent

An anticoagulant reversal agent can be administered as a therapeuticdrug in order to reverse partially or fully the effect of ananticoagulant. Restoration of the capacity of the blood to clot can belife-saving, for example, a patient who takes an anticoagulant and isexperiencing a severe injury can be treated with a reversal reagent forrestoration of blood clotting capacity and prevention of excessive bloodloss.

“Open” and “Closed” Devices

An “open” device has a channel to the outside from an interior chamber,allowing for direct access of blood into the chamber. A “closed” devicehas no exterior channels, and is filled before being sealed to theoutside except for small holes associated with sensors, electrodes,LEDs, and other elements utilized in assessing clotting within thechamber. The ‘open version’ of the microfluidic system is designed sothat the chip can be inserted into its packaging first, be heated up,but still be accessible (open). A sample can be loaded into the systemthrough wetting of the side port, with the chip residing already in itspackaging. The sample will be drawn into the chip by capillary forces.

The ‘closed version’ of the microfluidic system will not be accessibleto the user after it is placed into its packaging. A sample has to beloaded into the system prior to placement of the chip into its packagingthrough wetting of or pipetting into one of the back side ports.

II. Device

Embodiments of the microfluidic chip and its reader are shown in FIGS.2-10, with the system including both parts reflected by FIG. 11. Asindicated in FIGS. 2A and 2B, microfluidic chips 10 are fabricatedthrough anisotropic wet etching of silicon wafers and subsequent thermaloxidation, isotropic wet etching of PYREX® (a clear,low-thermal-expansion borosilicate glass) wafers, sputter deposition ofthin metal films onto both wafers through stencils, anodic bonding ofsilicon and PYREX® wafers, and subsequent separation of single chips bywafer dicing.

The microfluidic chips can be fabricated by alternative means, using anymethod that is suited to generate microfluidic structures and anynegatively charged material that is suited to activate the bloodclotting cascade.

The cross-sectional dimensions and geometries of the chambers 12 a and12 b and the channel 14 connecting the chambers can be modified, and thenumber of chambers can be varied. The surface to volume ratio willoverall influence the clotting time. Access to the chambers is direct(“closed”, FIG. 2B), prior to sealing of the chamber device, or via aside channel 16 that allows access from the exterior of themicrochambers.

As shown by FIG. 3A and 3B, the chips are designed to enable:

-   -   Heating through backside resistance or electrical heater        structure (on underside of chambers 12 a and 12 b)    -   Temperature measurement for heating control through top side        (outer) thermistor 18    -   Clotting detection through air pressure measurements    -   Clotting detection through measurement of impedance across blood        sample through embedded electrodes 20 a and contact pads 20 b    -   Clotting detection through optical measurements

As shown in FIGS. 4A-4H, resistance structures 22 are deposited onto theback of a silicon wafer 24 (to form resistive heating structures 32,FIGS. 4G, 4H), onto the front side 26 of the silicon wafer (to formelectrodes 20 a, 20 b for impedance measurements and thermistors 18,respectively, at the floor of each chamber), and onto the front side ofthe PYREX® wafer (to form thermistors 18 on top of one or both chambers12 a, 12 b). The heater and the thermistor can be external to the“chip”, and can be integrated into the reader structure. If the walls ofthe cavity into which the chip is placed are of significantly greatermass than the chip, highly thermally conductive, and form an almostcomplete surround, then the cavity approximates a “black body” and thechip must come to thermal equilibrium with the cavity. If the chip is incontact with the cavity, or closely spaced, the equilibrium timeconstant can be very short. This can be established by pyrometermeasurement during development. This should reduce the complexity of thedisposable part of the system, and the cost. Connecting channel 14 andentry port 36 can be etched using potassium hydroxide (KOH) into thesilicon wafer 26.

The device in FIG. 2B, 3B (called ‘closed device’) has two entry ports28 a, 28 b etched through the silicon part 30. A sample can be pipettedinto one of the ports 28 a, 28 b before the device 10 is inserted into asealed packaging. The device in FIGS. 2a, 3b has one entry port 28 betched through the silicon part and one sideway entry port 16, realizedas channel etched into the PYREX® (called ‘open device’). In the casesof a silicon substrate, the surface wave structure can be directlyintegrated into the microfluidic design by well understoodmicrofabrication techniques. An open device chip (FIGS. 2A, 3A) can beinserted into a sealed packaging and/or reader first, with the edge withthe sideway port sticking out of the reader. Wetting of the sideway port16 will then result in sample being drawn into the chamber 12 a bycapillary action.

The combined heater/cooler control system and the thermistor can beexternal to the ‘chip’, and can be integrated into the reader structure.If the walls of the cavity, into which the chip is place, are ofsignificantly greater mass than the chip, highly thermally conductive,and form an almost complete surround, then the cavity approximates a‘black body’ and the chip must come to thermal equilibrium with thecavity. If the chip is in contact with the cavity, or closely space, theequilibrium time constant can be very short. This can be established bypyrometer measurement during development. This would reduce thecomplexity of the disposable part of the system and (hopefully) thecost.

Chip packages or “readers” as shown in FIGS. 5-10, provide electrical,optical and fluidic interfaces to the chip. A reader consists of abottom (FIGS. 5, 6, 9A, 9B) and a top (FIGS. 7, 8, 10A, 10B) part thatare manufactured by high precision 3D printing, molding, machining, orother fabrication processes. Both parts are joined and pressed againsteach other by locking metal dowel pins that fit into holes 56. Thereader can include means for display, storage of information, and acommunications capability.

FIGS. 5 and 6 show the bottom reader part 50 for the closed device chipfrom two different angles. Channels 52 a, 52 b, located in recess 54,inside the reader connect chambers 2 and 1, respectively, to the chipentry ports. On one side of the reader, a solenoid valve (not shown) canbe attached to the reader at channel ends 58 to control the connectionbetween the reader channel and a barbed tube connector that is screwedinto the bottom of the reader. On the opposing side, a pressure sensor(not shown) can be attached to the reader at holes 60 to monitor thepressure applied to the reader channel and chip entry port. Each entryport 62 a, 62 b is controlled/monitored by its own solenoidvalve/pressure sensor secured at holes 70 a, 70 b, 70 c, and 70 d. Thebottom reader part exhibits a recess 54 for the microfluidic chip. Smallvertical holes 64 a, 64 b hold pogo pins to contact the heater structureat the bottom of the chip. A hole 66 at the center of the reader partholds an IR LED chip in a metal can reader, which passes along lightpath 68.

The IR components can be molded or integrated into a “chipstrate” form.

Although shown as a single point optical measurement, a multipointoptical measurement could be used.

FIGS. 7 and 8 show the top reader part 80 for the closed device chipfrom two different angles. The large (for example, 5 mm) hole 82 in thecenter holds an IR photodiode chip (not shown) in a metal can readerthat directs light through hole 86. Three small (for example, less thanone mm) vertical holes 84 hold three pogo pins (not shown) to contactthree electrodes for impedance measurements (one common ground electrodefor both chambers and one counter electrode in each chamber), so thatimpedance measurements can be carried out in both chambers. Two othervertical holes 88 hold pogo pins to contact the thermistor on top of thechip. Other embodiments of these devices are known and readily availablefor the same function. IR LED and photodiode are placed so that theyinterrogate the 1 mm diameter center region of one chamber.

The chip reader itself is attached to the cover of a project box 100that contains electronic circuitry, valves, and pumps needed to performautomated measurements, as shown in FIG. 11. The project box 100 isconnected to a PC 102 where measurements are controlled by a LabViewprogram and processor 110, and results shown on monitor 104.

FIGS. 9A and 9B show the bottom reader part 120 for the open device chipfrom two different angles. Dowel pin holes 122 are used to secure thedevice. Channels 124 inside the reader connect chambers 2 and 1,respectively, to the chip entry ports. On one side of the reader, asolenoid valve (not shown) can be attached to the reader at channel endsto control the connection between the reader channel and a barbed tubeconnector that is screwed into the bottom of the reader. On the opposingside, a pressure sensor (not shown) can be attached to the reader atholes 126 a, 126 b to monitor the pressure applied to the reader channeland chip entry port. Each entry port is controlled/monitored by its ownsolenoid valve/pressure sensor secured at holes. The bottom reader partexhibits a recess 128 for the microfluidic chip. A hole 130 at thecenter of the reader part holds an IR LED chip in a metal can reader.

FIGS. 10A and 10B show the top reader part 140 for the open device chipfrom two different angles. The large (for example, 5 mm) hole 142 in thecenter holds an IR photodiode chip (not shown) in a metal can readerthat directs light through hole 144. Three small (for example, less thanone mm) vertical holes 146 hold three pogo pins (not shown) to contactthree electrodes for impedance measurements (one common ground electrodefor both chambers and one counter electrode in each chamber), so thatimpedance measurements can be carried out in both chambers. Two othervertical holes 148 hold pogo pins to contact the thermistor on top ofthe chip. IR LED and photodiode are placed so that they interrogate the1 mm diameter center region of one chamber.

Although described with reference to interrogation of both chambers withone beam, it is possible to interrogate both chambers using separatebeams. One beam is shown for illustration purposes only.

The chip reader is attached to the cover plate of a project box asdescribed above using screw holes 150, as shown in FIG. 11.

A. Surfaces for Activation of Blood Clotting

The microfluidic system is fabricated so that the introduced blood orplasma sample is in contact with a glass surface, the top part of themicrofluidic chip and/or the surface of the bottom part of themicrofluidic chip, formed of a material such as PYREX® or thermallyoxidized silicon, such as amorphous SiO₂, silicon oxide, and siliconnitride. The glass serves to activate the clotting cascade without useof additional chemical or biological reagents. Activation is eitherachieved through mere contact of the sample with the glass surface orthrough active movement (for example, through an externally applied airpressure pulse) of the sample along the glass surfaces inside themicrofluidic system.

The microfluidic system is fabricated so that the introduced blood orplasma sample is in contact with glass surfaces (or other negativelycharged surfaces), which serve to activate the clotting cascade withoutuse of additional chemical or biological reagents. Glass surfaces aregenerated through use of glass wafers and oxidized silicon wafers,respectively, for fabrication of microfluidic systems. Alternatively,glass surfaces can be realized through use of glass chips that areintegrated in reader parts that form a microfluidic system, throughdeposition of glass onto the inner surfaces of the microfluidic system(for example, through use of spin-on glass products) or throughintegration of small objects with glass surfaces (for example, glassmicrobeads) inside the microfluidic system. Additionally glass surfacescan be introduced by the oxidation of silicon surfaces.

B. Electrical Characteristics of Deposited Metal Thin Films

Deposited metal thin films can be formed of chromium adhesion layers(approximately 20 nm thick) and gold top layers (approximately 50 nmthick for inner electrodes for impedance measurements, 100 nm thick forthermistors and 150 nm thick for heater structures). Spring-loaded pogopins in the plastic reader were used to realize electrical contacts toall thin film electrodes on the microfluidic chip. Typical resistancesbetween two pins connected to either end of a metal film test structure,with approximate length of 2 mm and width of 1 mm, are 1.8 Ohm, withpins contacting metal films for heater structures, 2.7 Ohm, contactingmetal films for thermistors, and 22 Ohm, contacting metal films forinner electrodes for impedance measurements. The markedly higherresistance between inner electrode pins is likely due to a thinner goldlayer and possibly contact degradation during anodic bonding atapproximately 300° C. Heater structures and thermistors are depositedafter anodic bonding.

For measurement of temperature coefficients of resistances of depositedmetal thin films, a chip was used that exhibited resistor/thermistorstructures instead of open circuit electrodes for impedancemeasurements. The chip was inserted into its reader and heated up insidean oven. Electrical resistances of heater, thermistor and innerelectrode resistors were measured at different temperatures andtemperature coefficients of resistances a were calculated:

-   -   heater thin film: α=0.0016 K⁻¹    -   thermistors thin film: α=0.00115 K⁻¹    -   inner electrode thin film: α=0.000108 K⁻¹.

C. Internal Electrodes for Sample Positioning

Apart from impedance measurements, integrated electrodes can also beused to detect the presence of the analyte, such as fibrin, in themicrofluidic system and/or to track movement of the analyte, forexample, due to externally applied air pressure pulses. Such detectionand tracking can be used to initiate the analysis procedure once theanalyte is added to the microfluidic system, to position the analyte ata specific location within the microfluidic system, and to move itrepeatedly back and forth between defined locations, respectively.

Although exemplified with reference to two chambers and two electrodes,multiple electrodes can be used to confirm filling of multiple chambers,at the site of measurement of clotting or at a point prior to thechamber where the clotting is measurement, such as closer to the inlet.

Repeated movement of whole blood or blood plasma along glass surfacescan be applied to increase activation of the blood, to accelerate bloodclotting and/or to decrease measurement times.

D. Integration of Filter Structures

Mechanical filter structures can be integrated into the microfluidicchip, so that only blood plasma is arriving at the analysis chambers.The filters can be realized as array of micropillars or as microchannelsetched into silicon or glass. This way, plasma (without the use of ananticoagulant such as sodium citrate or EDTA) can be produced in situand very quickly tested in the same manner as whole blood, without redblood cell interference in the analysis. The micropillar arrays can bearranged in offset patterns to inhibit red blood cell transit, whileminimizing the probability of “plugging” an excessive fraction of theavailable channel cross section.

E. Means for Clotting Detection

A variety of modalities can be applied to determine blood clottingtimes.

Viscosity

The viscosity of the blood can serve as a measure to characterizeclotting times. Two general principles may be applied to yield a director indirect measure for the viscosity. The sample can be moved through achannel with known geometry. Viscosity can be measured by tracking therate of penetration through a “long” channel either optically or byimaging or multiple beam “check points” or electrically by multipleelectrode impedance sensors. The viscosity may be measured indirectly,for example through measurement of the distance the sample has travelledinside a channel during a specific time interval, the sample volume thathas been displaced during a specific time internal, or the change indriving force during a specific time interval (for example, if thesample is moved by a pressurized volume of trapped air, the change inair pressure can serve as an indirect measure for sample volumedisplacement). Alternatively, objects can be moved through the bloodsample, for example, driven through electrostatic or magnetic forces.Tracking of the object movement can yield an indirect measure for thesample viscosity.

The viscosity of the analyte can be detected through movement of theanalyte inside the microfluidic system through a pressurized, entrappedair volume. Air can be pressurized, for example, through electric airpumps that are connected to the microfluidic system. Pressurized air canbe entrapped through closure of solenoid valves connected to themicrofluidic system. Decreasing pressure of the entrapped air at oneentry port of the chip indicates movement of the analyte. Knowledge ofthe geometry of the microfluidic system and the magnitude of the appliedpressure allows calculation of analyte viscosity and detection ofviscosity changes (for example, a viscosity increase due to clotting incase of blood).

Clot detection by viscosity monitoring involves measuring differentialpressure across an on-chip inlet and outlet, connected to fluidic portson the reader (made air/fluid tight using o-ring seals).

The viscosity of the analyte can be detected through movement of theanalyte inside the microfluidic system through a pressurized, entrappedair volume. Air can be pressurized, for example, through electric airpumps that are connected to the microfluidic system. Pressurized air canbe entrapped through closure of solenoid valves connected to themicrofluidic system. Decreasing pressure of the entrapped air at oneentry port of the chip indicates movement of the analyte. Knowledge ofthe geometry of the microfluidic system and the magnitude of the appliedpressure allows calculation of analyte viscosity and detection ofviscosity changes (for example, a viscosity increase due to clotting incase of blood).

Impedance

Clotting of a sample can be related to its electrical impedance, or itscomplex resistance when an electric current or voltage is applied. Theelectrical impedance results from ohmic resistances as well ascapacitive components of the sample. The electrical impedance can, forexample, be measured through electrodes that are directly integrated inthe microfluidic chip or integrated in the reader that forms, togetherwith other parts, a microfluidic system. Electrodes can be partially indirect contact with the sample or separated from the sample only througha thin insulator (with a thickness ranging from nanometers up tohundreds of micrometers).

Electrodes and conductor lines may be formed as patterned thin metalfilms that are deposited onto substrates forming the microfluidicsystem. Electrodes and conductor lines can also be realized throughintegration of patterned metal sheets or films that are inserted andsandwiched between reader parts. If semiconductor wafers are used tofabricate microfluidic chips, the semiconductor material itself cancontain integrated electrodes fabricated by diffusion, implantation,etching, micro machining, or any combination of appropriate techniquessimilar to the techniques used in integrated circuit or other microdevice production. The integrated electrodes can be used to measuredirectly electrical properties of the analyte (for example, resistance,capacitance, impedance). Suitable electronic circuits may also be usedto translate analyte changes (and related changes in electricalproperties) into measureable electric voltages, currents, frequencies orother suitable parameters. Parts can also be inserted duringconstruction by 3D printing, or integrated directly. For example, aresistor heating element would be fabricated by a doped channel in asemiconductor substrate (by ion beam implantation, for example).

Clot detection by impedance monitoring is accomplished by inserting thechip into the reader, making contact between gold pads (connected to theon-chip electrodes) and Pogo pins in the reader from which theelectrical signal is read by an LCR meter, for example, or any otherappropriate measurement system.

For detection of clotting through measurement of impedances, a sample isloaded into the chip, the chip is inserted into its reader, and the pogopins connecting to the internal impedance electrodes connected viaKelvin clip leads to a QuadTech 1920 LCR meter. The magnitude and thephase of the complex impedance of the blood sample were recorded at 15second intervals. Measurements at 100 Hz, 1 kHz, 10 kHz, and 100 kHzshowed characteristics peaks or plateaus of either the magnitude or thephase or both. The peaks or plateaus indicated a measure for theclotting time.

Referring to FIGS. 3A-3B, for impedance measurements, the external LCRmeter applies an AC voltage (20 mV RMS) between the two electrodes 20 b,20 a on each side of the chamber 12 a, and measures the electricalcurrent between the electrodes. The magnitude and phase of impedance arethen computed and the clotting calculated.

Optical Properties

Optical properties of the sample can be related to clotting events.Light with wavelength in the range of 500 nm to 10,000 nm (preferably1,300 nm) can be used to illuminate the sample through the microfluidicand chip reader. The following parameters can be used to track clotting:transmitted, reflected and scattered light. If a coherent light sourceis used, polarization may be used as additional parameter.

Clot detection by IR transmission is performed by inserting the chipinto the reader and measuring infrared transmission across the thicknessof the chip (through the glass, fluid-fill chamber, and underlyingsilicon). This is accomplished via placement of an IR source (LED) abovethe chip and photodiode detector aligned immediately below.

For optical clot detection, IR LED and photodiode are inserted intotheir reader parts as described above. A blood sample is pipetted intothe chip and the chip placed into the reader. The sample is continuouslyilluminated with IR light at 1,300 nm. At time intervals of typically100 ms, the voltage drop across a 1 MOhm resistor caused by thephotocurrent of the photodiode was recorded. Voltages typically measuredseveral volts. Clotting of the sample caused the transmitted light andthe photocurrent to vary over time. Characteristic peaks of thetransmitted light curve indicated a measure for the clotting time.

The continuous illumination measurement is presented as a simpleillustration. More sophisticated measurement techniques may be used. Forexample, if the IR emitter were illuminated for 50% duration, at arepetition rate of 1 khz, and a synchronous detector were used toprocess the photodetector output, followed by a 1 second integrationperiod, the signal to noise ratio in the above example could be improvedby as much as thirty-fold. In addition, active signal processing wouldallow processing of much smaller signals, permitting a relatively lowimpedance termination of the photodetector, lowering the intrinsicnoise, and canceling drift. Ambient electrical noise sensitivity wouldbe substantially reduced.

Acoustic Properties

Measurement of sound propagation in the sample or along the samplesurface can serve as an additional measure for clotting. Externalultrasound transducers can be used to measure the time it takesultrasound to travel through the sample. Additionally, surface acousticwave devices can be used to measure acoustic properties of the sampleand to detect clotting.

III. Methods of Making Devices

In addition to standard processes such as photolithography, specialtechnologies such as anodic bonding or potassium hydroxide anisotropicwet etching of silicon wafers can be applied to form microsystems. Apartfrom standard ultraviolet light lithography, techniques such as directlaser writing microablation or erosion, electron beam lithography, orfocused ion beam milling can be used to define micro- or nanometer-sizedstructures. Soft lithography is a related way to fabricate microfluidicsystems and is based on generation of microstructures or -patterns, forexample, through standard photolithography techniques, and subsequentuse of these patterns in molding/casting processes. Elastomeric materialsuch as polydimethylsiloxane (PDMS) are typically used for generation ofmicrofluidic system by soft lithography. Structured films generated bysoft lithography can be attached to each other or to any otherstructured or non-structured substrate to form complex microfluidicsystems. Furthermore, other technologies such as drilling, milling,molding, or 3D printing, may be used alone or in combination with othermicro-/nanotechnologies to fabricate microsystems.

IV. Sample Collection

Blood Collection

In most cases, individuals to be tested will present at a clinic or ahospital, possibly with unknown status as to treatment withanticoagulants. Blood can be obtained by the use of a syringe, alancelet, or directly from a blood containing line. Due to the use ofthe alternative clotting pathway in which clotting is activated using aglass type surface, the blood may contain anticoagulants such aswarfarin, heparin, low molecular weight heparin, factor IIa inhibitors,factor Xa inhibitors, and other factor inhibiting or factor impairedblood.

Warfarin and related 4-hydroxycoumarin-containing molecules decreaseblood coagulation by inhibiting vitamin K epoxide reductase, an enzymethat recycles oxidized vitamin K₁ to its reduced form after it hasparticipated in the carboxylation of several blood coagulation proteins,mainly prothrombin and factor VII. Warfarin does not antagonize theaction of vitamin K₁, but rather antagonizes vitamin K₁ recycling,depleting active vitamin K. Thus, the pharmacologic action may always bereversed by fresh vitamin K. When administered, these drugs do notanticoagulate blood immediately. Instead, onset of their effect requiresabout a day before remaining active clotting factors have had time tonaturally disappear in metabolism, and the duration of action of asingle dose of warfarin is 2 to 5 days. Reversal of warfarin's effectwhen it is discontinued or vitamin K₁ is administered, requires asimilar time.

Heparin is a compound occurring in the liver and other tissues thatinhibits blood coagulation. A sulfur-containing polysaccharide, it isused as an anticoagulant in the treatment of thrombosis. Low molecularweight heparin, a more highly processed product, is useful as it doesnot require monitoring by aPTT coagulation parameter (it has morepredictable plasma levels) and has fewer side effects. However, inemergency bleeding situations the ability to monitor LMWH is asignificant unmet clinical need as no point of care assay is clinicallyaccepted for LMWH anticoagulant monitoring.

Drugs such as rivaroxaban, apixaban and edoxaban work by inhibitingfactor Xa directly (unlike the heparins and fondaparinux, which work viaantithrombin activation).

Another type of anticoagulant is the direct thrombin inhibitor. Currentmembers of this class include the bivalent drugs hirudin, lepirudin, andbivalirudin; and the monovalent drugs argatroban and dabigatran.

The sample can be tested as blood or as plasma. Plasma can be preparedby filtration or centrifugation. Additionally, additional glass surfacearea can be added to one or more of the microfluidic channels by theintroduction of glass beads into the channel using for example a doubledepth chip or in-channel bead packing.

Other Biological Samples

The device can be used with other types of samples that are activatedwith exposure to glass.

V. Methods of Use

The samples are collected and administered into the device. The meansfor determining clotting are started as the sample is placed into thedevice. Results are compared to standard results for uncoagulatedsamples, typically from pooled plasma or pooled blood, or by referenceto the clotting time at initiation of treatment, as in the case where anindividual is administered anticoagulant, or a therapeutic to neutralizethe anticoagulant and restore more normal blood clotting.

The present invention will be further understood by reference to thefollowing non-limiting examples.

EXAMPLE 1 Demonstration of On-chip Heating

A chip was used to demonstrate the effect of the integrated heater (orintegrated heater/cooler; preferably a solid state heat pump or ‘Peltiercooler’) structure. A 12 V DC voltage was applied to the heater resistoron the back of the silicon part of the microfluidic chip. Resistances ofthermistors were measured inside each chamber (inner thermistors, on thefront silicon surface) and on top of each chamber (outer thermistors, ontop of the PYREX®) before application of a heater voltage and duringheating. Local temperatures increase due to heating were calculatedusing measured resistances and temperature coefficients of resistancesas reported earlier. Room temperature was approximately 27° C. Averagelocal temperature increases after approx. 2 min of unregulated heatingwere:

-   -   outer thermistors: ΔT=20.3 K    -   inner thermistors: ΔT=23.8 K.

EXAMPLE 2 Measurement of Blood Clotting

Materials and Methods

Blood was harvested from a patient using commercially available lancingdevices. 10 μL of blood were obtained and pipetted into an Eppendorftube. Saline was used as a buffer solution. The blood sample was mixedin the Eppendorf tube through up and down pipetting five times with oneof the following reagents:

-   -   1 μL of buffer solution (called sham control),    -   1 μL of buffer solution containing the anticoagulant edoxaban at        a concentration of 300 ng/mL,    -   1 μL of buffer solution containing the anticoagulant edoxaban        and the anticoagulant reversal agent PER977, both at a        concentration of 300 ng/mL.

Edoxaban is a commercially available anticoagulant. PER977(ciraparantag) is an investigational drug that is designed to reversethe effect of edoxaban. Immediately after mixing, 2.5 μL of each bloodsample was pipetted into a closed device chip. The chip was insertedinto its reader, and both IR light and impedance measurements wereimmediately recorded at room temperature (approximately 27° C.). Heatingof the blood sample were omitted.

Results were obtained both by IR and viscosity impedance.

Results

As evident from FIGS. 12A-12F, IR (FIGS. 12B, 12D, and 12F) andimpedance measurements (12A, 12C, and 12E) correlate well with eachother. Both measurements show for the sham control a characteristic peakaround 2 minutes that is indicative of the sample clotting time.Addition of the anticoagulant edoxaban shifts this peak to approximately4 minutes. In addition to the peak, the edoxaban curves show acharacteristic local minimum around 12 minutes. Addition of theanticoagulant reversal agent PER977 to a blood sample containingedoxaban shifts the peak in each curve back to 2 minutes and suppressesthe occurrence of a local minimum around 12 minutes. These measurementsindicate the clotting-delaying effect of edoxaban and the reversal ofthis effect through additional administration of PER977.

Modifications and variations of the devices, systems and methods of usethereof will be evident to those skilled in the art from the foregoingdetailed description and are intended to come within the scope of theappended claims.

1-14. (canceled)
 15. A test microchip for measuring clotting in a bloodor plasma sample, the test microchip comprising: an inlet for the bloodor plasma sample, the inlet communicating with one or more microchannelshaving a length between tens of microns and millimeters, eachmicrochannel comprising one or more test chambers, each microchannelhaving a defined volume between nanoliters and milliliters andconfigured to draw the blood or plasma sample into the one or more testchambers by passive capillary action, the one or more microchannelscommunicating with an outlet and each of the one or more microchannelscomprising at least one anionically charged surface which activatesclotting of the blood or plasma sample upon entry of the blood or plasmasample into the one or more microchannels or test chamber, wherein theanionically charged surface does not include chemical agents activatingclotting, and wherein changes in electrical impedance indicative of clotformation can be measured.
 16. The test microchip of claim 1, whereinthe test microchip includes integrated electrodes which allows changesin the electrical impedance in the blood or plasma to be measured. 17.The test microchip of claim 2, wherein the test microchip comprises asingle microchannel.
 18. The test microchip of claim 2, wherein theintegrated electrodes are positioned in the test chamber so theintegrated electrodes will be in direct contact with the blood or plasmasample introduced into the test chamber.
 19. A microassay device formeasuring clotting in a blood or plasma sample from an individual, thedevice comprising a test microchip, the test microchip comprising: aninlet for the blood or plasma sample, the inlet communicating with oneor more microchannels having a length between tens of microns andmillimeters, each microchannel comprising one or more test chambers,each microchannel having a defined volume between nanoliters andmilliliters and configured to draw the blood or plasma sample into theone or more test chambers by passive capillary action, the one or moremicrochannels communicating with an outlet and each of the one or moremicrochannels comprising at least one anionically charged surface whichactivates clotting of the blood or plasma sample upon entry of the bloodor plasma sample into the one or more microchannels or test chamber,wherein the anionically charged surface does not include chemical agentsactivating clotting, and wherein changes in electrical impedanceindicative of clot formation can be measured, wherein the test microchipis inserted into a reader, the reader comprising a detector whichdetermines changes in electrical impedance in the blood or plasma sampleto measure clotting time, and a temperature control regulating thetemperature of the test chamber, wherein the detector is configured tooutput the measured clotting time from the time of activation of thesample to the time of change in the electrical impedance in the testchamber indicative of clotting.
 20. The microassay device of claim 5,wherein the output of the reader is provided on a display.
 21. Themicroassay device of claim 5, wherein the reader comprises an integratedheater for controlling the temperature of the test chamber in theinserted test microchip.
 22. The microassay device of claim 5, whereinthe detector comprises a measurement system for reading an electricalsignal to detect a change in electrical impedance in the sample in thetest chamber of the inserted test microchip.
 23. A method for measuringclotting time comprising the steps of applying a blood or plasma sampleto the test microchip of claim 1 in a reader for measuring clotting inthe blood or plasma sample, wherein the reader comprises a detectorwhich determines changes in electrical impedance in the blood or plasmasample to measure clotting time, and a temperature control regulatingthe temperature of the test chamber, wherein the detector is configuredto output the measured clotting time from the time of activation of thesample to the time of change in the electrical impedance in the testchamber indicative of clotting; and obtaining outputting of the clottingtime.
 24. The method of claim 9, wherein the change in electricalimpedance is measured by applying an AC voltage to the blood or plasmain the test chamber.